The long term goal of this project is to understand the pathogenesis of Venezuelan equine encephalitis virus (VEE) in molecular terms. In rodent models as well as in humans and equines, the virus first attacks lymphoid and myeloid tissues. Subsequently, VEE invades the central nervous system (CNS) and infects neurons and glial cells with consequent development of encephalitis. Under the auspices of the current grant, the tools required for a molecular genetic approach to gain insight into the details of this disease process have been developed. These include 1) a full-length cDNA clone of wild-type VEE, configured so that in vitro transcription with T7 polymerase yields infectious VEE RNA, and 2) isogenic clones containing single-step, attenuating mutations. The availability of these molecularly cloned, single-step VEE mutants not only provides the means of defining the specific stages of an important alphavirus induced disease, but they also offer a unique opportunity to explore fundamental mechanisms of viral pathogenesis. The premise of the approach is that single-site mutations which produce an avirulent phenotype are inhibited in their ability to progress beyond a certain point in the disease process. Therefore, determining the point(s) at which the mutants are inhibited will ultimately define critical steps in VEE pathogenesis for both the lymphotropic and neurotropic aspects of the disease. The inhibition of mutant replication in vivo will be studied further by determining the basis for the growth restriction in specific cell types of the target organs. This win be accomplished by 1) identifying cell types in vivo which are susceptible to wild-type infection but resistant to infection by a given mutant, 2) reproducing the differential restriction in cell culture, and 3) using the cell culture system to determine the basis for the restriction. Because the mutants and wild-type differ at only a single residue, it is likely that the basis of the cell culture restriction will also be the basis for restriction in the analogous cells in vivo. It will be determined whether lymphotropic involvement is a prerequisite for invasion of the CNS by VEE, and if so, the nature of this requirement. Finally, the effects of VEE and lymphotropic VEE mutants on host immune function will be examined. The project will provide new and significant information concerning the pathogenesis of an important human and veterinary pathogen. Moreover, the information gained from the study of VEE will have general implications for other lymphotropic and neurotropic viruses.